Imprinting represents a curious defiance of normal Mendelian genetics. Mammals inherit two complete sets of chromosomes, one from the mother and one from the father, and most autosomal genes will be expressed equally from maternal and paternal alleles. Imprinted genes, however, are expressed from only one chromosome in a parent-of-origin dependent manner. Because silent and active promoters are present in a single nucleus, the differences in activity cannot be explained by transcription factor abundance. Thus the transcription of imprinted genes represents a clear situation in which epigenetic mechanisms restrict gene expression. Therefore imprinted genes are good models for understanding the role of DNA modifications and chromatin structure in maintaining appropriate patterns of gene expression. Further, because of parent-of-origin restricted expression, phenotypes determined by imprinted genes are not only susceptible to mutations of the genes themselves but also to disruptions in the epigenetic programs controlling regulation. Thus imprinted genes are frequently associated with human diseases, including disorders affecting cell growth, development, and behavior. Our Section is investigating a cluster of genes on the distal end of mouse chromosome 7. The syntenic region in humans on chromosome 11p15.5 is conserved in genomic organization and in monoallelic expression patterns. Especially, we are focusing on the molecular basis for the maternal specific expression of the H19 gene and the paternal specific expression of the Igf2 gene. Loss of imprinting mutations in these two genes is associated with Beckwith Wiedemann Syndrome (BWS) and with Wilms tumor. Expression of both H19 and Igf2 is dependent upon a shared set of enhancer elements downstream of both genes. We have identified a 2.4 kb ICR (for Imprinting Control Region) upstream of the H19 promoter. Using conditional deletion and insertional mutagenesis we have identified three functions associated with this element. First, this element acts to distinguish the parental origin of any chromosome into which it is inserted. Specifically, the CpGs within this region become hypermethylated upon paternal inheritance. Second, this element functions as a CTCF-dependent, methylation-sensitive transcriptional insulator. By reorganizing the long-range interactions of nearby promoter and enhancer elements, this insulator is able to direct parental-specific activation of nearby genes. Finally, this ICR also acts as a developmentally regulated silencer element when paternally inherited. Specifically, the methylated ICR induces changes in chromatin structure of neighboring sequences that impacts gene expression. Our current goals are to identify and characterize the protein factors and non-coding RNAs that interact with the ICR and establish the chromatin structures associated with the maternal and paternal chromosomes. We are addressing these issues both in germ cells, where the imprints are established, and in somatic tissues where expression of Igf2 and H19 are most critical for normal, healthy cell function. We are also working to establish mouse models that mimic the Beckwith Wiedeman syndrome phenotypes associated with loss of imprinting at the Igf2/H19 locus in humans. Most recently we have demonstrated defects in muscle cell differentiation and in muscle regeneration in cells where Igf2/H19 imprinting is disrupted. We have demonstrated that even a <2-fold increase in Igf2 expression will result in large-scale disruption in cell cycle regulation by hyperactivation of the MAPK pathway. In addition, decreased expression of H19 disrupts normal regulation of p53 in muscle cells so that they can no longer respond to Wnt stimulation and therefore do no undergo normal hypertrophy. Thus loss of imprinting of both H19 and Igf2 genes are relevant to overgrowth phenotypes in BWS We are now characterizing cardiac dysfunction phenotypes in these mutant animals. This disease phenotype seems entirely associated with overexpression of Igf2 and H19 plays minimal or no role. Moreover, the relevant overexpression occurs early in development and in circulating Igf2 peptide derived from skeletal muscle and liver. These early embryonic changes are enough to explain the disease progress in adults. Thus therapies must target very early development to prevent disease initiation Igf2 encodes a peptide mitogen whose biochemistry is well understood. However, H19 encodes a long non-coding RNA and its biochemical roles remain unclear. We have generated novel mouse models with small mutations in the H19 RNA coding regions that delete putative miRNA encoding sequences and also putative let7 miRNA binding sites and will determine if these sequences play any role in the H19 functions described above. We are also generating a novel mouse model where H19 RNA is tagged with an aptamer that mimics biotin. We will use this tool to identify proteins that interact with H19. A second research goal is to generate mouse models for cardiac arrhythmias. We first focused on uncovering the biological function of the imprinted Kcnq1 gene, located just upstream of Igf2. More recently, we have generated mouse models for Calsequestrin2 deficiency. We demonstrated that calsequestrin2 is not essential for cardiac calcium ion storage, which can be maintained by an expansion of the sarcoplasmic reticulum (SR) volume and surface area. Rather, the primary function of calsequestrin appears to be the regulation of the SR calcium ion release channel during conditions of beta-adrenergic stimulation. The loss of calsequestrin2 thus results in premature calcium ion release from the SR, leading to voltage changes that result in premature contraction of cardiomyocytes and thus arrhythmia. The validity of this mouse model has been recently confirmed by demonstration that drugs that we used to successfully ameliorate the mouse arrhythmias were highly effective in pilot studies on human patients. In the past two years, we have demonstrated that the arrhythmias associated with calsequestrin2-deficiency worsen significantly with age. Through genomic analyses we conclude that this is because mitochondrial function is not appropriately upregulated in mutant mice. These results suggest new therapeutic targets for human patients. We have recently completed analyses of conditional alleles of calsequestrin 2. Casq2-deficient mice closely phenocopy the human disease. That is mice show normal heart function (but reduced heart rate) under basal conditions but develop polymorphic ventricular tachycardia (CPVT) in response to stress. Phenotypic analyses of these mice show that the CPVT phenotype is independent of developmental history. That is, the presence of arrhythmia depends on the status of Casq2 at the time of analysis with minimal influence by the hearts developmental history in regards to Casq2 gene function. Moreover, our data indicate that CPVT phenotype is dependent upon concurrent loss of Casq2 peptide in both the cardiac conduction system (CCS) and in working cardiomyocytes. The practical significance of this finding is that therapies that rescue Casq2 only in the CCS may be sufficient to prevent CPVT. In contrast to the CPVT phenotype, heart rate phenotypes are dependent only on the loss of Casq2 in the CCS. More interestingly, heart rates are dependent upon CCS developmental history. That is, heart rates are determined by two factors: 1) the status of Casq2 at the time of analysis and 2) CCS developmental history in regards to Casq2 gene function. Altogether, our data indicate that the relationship between heart rate and CPVT is complex but support the idea that reduced basal heart rate is a central contributor to increased risk of stress-induced arrhythmias in Casq2-deficient hearts.